Astroviruses are small, spherical, non-enveloped, positive-sense RNA viruses (28-30 nm) that cause enteric diseases in mammalian and avian species. Astroviruses of chickens have been implicated in growth depression problems, including runting stunting syndrome. Astrovirus infection is a global problem affecting broiler chicken production, which results in financial losses from increased culling, poor feed conversion and lower uniformity at slaughter with concomitant increased costs from treatment.
To date, two different astrovirus species have been detected in chickens, avian nephritis virus (ANV), and a novel astrovirus, named chicken astrovirus (CAstV). Establishing the importance of astrovirus infections in broiler growth problems has been difficult due to the absence of convenient diagnostic tests. Whilst electron microscopy (EM) can demonstrate avian astroviruses in diagnostic samples, this method relies on observing the star-like morphology which is in apparent in some types of astrovirus including ANV. In addition, EM is not suited to high sample throughput and lacks sensitivity. Isolating astroviruses in cell culture presents difficulties as they grow poorly and are often outgrown by reoviruses and adenoviruses, which also occur commonly in enteric samples. Antigen-detecting diagnostic tests including fluorescent antibody tests performed with cryostat tissue sections or tissue impression smears and antigen capture ELISA have not been developed for CAstVs due to the absence of virus-specific antisera.
A number of publications, for example WO2007/077464; Avian Dis., Vol. 50, 2006, pages 397 to 404, Pantin-Jackwood, M. J., Spackman, E., and Woolcock, P. R., “Molecular characterization and typing of Chicken and Turkey Astroviruses Circulating in the United States: Implications for Diagnostics”; Avian Dis., Vol. 51, 2007, pages 681 to 684, Day, J. M., Spackman, E., and Woolcock, P. R., “A Multiplex RT-PCR Test for the Differential Identification of Turkey Astrovirus Type 1, Turkey Astrovirus Type 2, Chicken Astrovirus, Avian Nephritis Virus and Avian Rotavirus”; and Avian Pathol., Vol. 38, 2009, pages 21 to 29, Todd, D., Smyth, V. J., Ball, N. W., Donnelly, B. M., Wylie, M., Knowles, N. J. and Adair, B. M. “Identification of Chicken enterovirus-like viruses, duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses” describe the use of Reverse Transcriptase Polymerase Chain Reaction (PCR) methodologies to detect enteric viruses in avians. However, generally these tests, for example as described in the publications of Pantin-Jackwood M. J et al and Todd D. et al, use primers which cannot distinguish between different avian astroviruses and therefore gene sequencing is necessary to identify particular virus types within a sample. Whilst the publication of Day J. M. et al identifies the problem that RT-PCR tests for avian astroviruses generally cannot distinguish between different astroviruses and discusses the use of a multiplex RT-PCR test to enable identification of particular virus types, this publication highlights that due to limited sequence information for virus types such RT-PCR tests may only be able to detect a limited number of viral strains within a virus type. As discussed herein, the primers determined by the present inventors provide for improved detection of CAstVs in a sample.
In the RT-PCR tests previously described, the primer pairs utilised target the RNA polymerase (ORF1B) sequence.
The detection of CAstV from avian samples is a developing field which has been limited both by the low availability of sequence information and the high degree of sequence diversity often displayed by RNA viruses. The sequence diversity observed has made it difficult to identify conserved regions on which to base the design of oligonucleotide primers for use in RT-PCR tests for the detection of CAstV. The development of a test with reliable breadth to allow universal detection of CAstV and increased sensitivity is still required.